Neurodegenerative diseases comprise a large group of pathologies caused by metabolic changes in brain cells, loss of synapses and other compartments of neurons, and finally neuronal death. For review see Neurodegenerative diseases: From Molecular Concepts to Therapeutic Targets. Editors: R. von Bernhardi, N.C. Inestrosa, Nova Publishers, 2008. This group of diseases includes Mild Cognitive Impairment (MCI), Alzheimer's disease (AD), Lewy Body dementia, Parkinson's disease (PD), Huntington's disease (HD), frontotemporal dementia (FTD), vascular dementia, HIV Associated Neurocognitive Disorders (HAND), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), prion diseases, different ataxias, and others. Due to increased lifespan, neurodegenerative diseases have become very common in developed countries. There are about 6 million people living with AD in the US only, 70-80 million people are in the risk group and $148 billion is spent in the US for AD patient treatment and care. Drug development and successful treatment of AD and other neurodegenerative diseases are significantly complicated by the absence of effective methods for their early diagnosis and monitoring. Development of effective diagnostic methods is further complicated by the strong brain potential to compensate for the dysfunction and loss of neurons over a long period of time. This results in late clinical manifestation of disease symptoms when treatment cannot be very successful due to serious morphologic changes in the brain including the massive loss of neurons. Thus, diagnostic methods based on detection of early events in the disease development are particularly desirable.
Neurodegenerative diseases are characterized by neuronal death in different disease-specific areas of the brain. However, the neuronal loss is a relatively late event, typically following synaptic dysfunction, synaptic loss, neurite retraction, and the appearance of other abnormalities such as axonal transport defects. See, e.g., Bredesen, Molecular Neurodegeneration 2009, 4:27; Siskova et al., Am J. Pathol. 2009, 175(4):1610-21; Kielar et al., Hum Mol Genet. 2009, 18(21):4066-4080; Nimmrich and Ebert, Rev Neurosci. 2009, 20:1-12; Bellizzi et al., J Neuroimmune Pharmacol. 2006, 1:20-31; Milnerwood and Raymond, J. Physiol. 2007, 585:817-831; Waataja et al., J. Neurochem. 2008, 104:364-375; Fuhrmann et al., J. Neurosci. 2007, 27:6224-6233; Yoshiyama et al., Neuron. 2007, 53:337-351; Wishart et al., J Neuropathol Exp Neurol. 2006, 65:733-739; Gylys et al., Neurochem Int. 2004; 44:125-131; Conforti et al., Trends Neurosci. 2007, 30:159-166; Baloyannis et al., J Neurol Sci. 2006, 248:35-41; Diaz-Hernandez et al., FASEB J. 2009, 23:1893-1906; Spampanato et al., Neuroscience 2008, 157:606-620; Wade et al., Brain Res. 2008, 1188:61-68; Centonze et al., J. Neurosci. 2009, 29:3442-3452; Wegner et al., Neurology. 2006, 67:960-967; Dupuis and Loeffler, Curr Opin Pharmacol. 2009, 9:341-346; Revuelta, et al. Am J Alzheimers Dis Other Demen 2008 23: 97-102. Numerous studies are devoted to description of axon destruction with shedding of membrane-enclosed “axosomes”, axon, dendrite and spine pruning, and disassembly of synapses (Goda, Davis, Neuron 2003, 40:243-264; Eaton, Davis, Genes Development, 2003, 17:2075-2082; Koiral, Ko, Neuron, 2004, 44:578-580; Bishop et al., Neuron, 2004, 44:651-661; Low, Cheng, Phil. Trans. R. Soc. B 2006 361, 1531-1544).
Currently, diagnosis of AD and other forms of dementia is based on analysis of the patient's cognitive function. As mentioned above, due to effective compensatory mechanisms in the brain, the decrease of cognitive function is usually registered when a disease is in its later stages and fewer treatments are available. New imaging techniques, which are becoming increasingly popular (e.g., positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), multiphoton imaging, magnetoencephalography (MEG), electroencephalography (EEG) etc.), are helpful, however, they are currently not sufficiently sensitive and specific for detecting early stages of a disease before major morphological changes occur (Mucke, Nature, 2009, 461:895-897; Mistur et al., J. Clin. Neurol., 2009, 5:153-166; Miller, Science, 2009, 326:386-389; Perrin et al., Nature, 2009, 461: 916-922).
The existing diagnostic molecular tests for AD and other forms of dementia can be divided into two groups. The first group is based on analysis of single nucleotide polymorphisms (SNP), which is helpful for predicting a higher risk of a disease but not for diagnostics (Bettens et al.; Hum Mol Genet. 2010, 19(R1):R4-R11). The second group uses analysis of proteins involved in AD pathogenesis or brain-specific proteins, like neural thread protein (NTP), in bodily fluids (Schipper, Alzheimer's & Dementia. 2007, 3:325-332). However, these tests are not sufficiently sensitive and specific. Recently published data have demonstrated high sensitivity of AD detection by measuring concentrations of three protein biomarkers (beta-amyloid protein 1-42, total tau protein, and phosphorylated tau181P protein) in the cerebrospinal fluid (CSF) (Meyer et al., Arch Neurol. 2010, 67:949-956). The high invasiveness of the CST collection procedure makes such tests impractical and challenging for everyday clinical use.
Metabolic changes occurring in neurodegenerative diseases cause the destruction of spines, dendrites, axons, and synapse loss, and the latter, most likely, induces neuronal death (Bredesen, Molecular Neurodegeneration 2009, 4:27). Similar processes happen during embryonic brain development. Numerous neurons are trying to establish intercellular contacts, those neurons that do it successfully survive, and other neurons die (Butts et al., Cell Death Differ. 2008, 15:1178-1186; Enokido and Hatanaka, Gan To Kagaku Ryoho. 1994, 21:615-620; Gasic and Nicotera, Toxicol Lett. 2003, 139:221-227).
Axon destruction with shedding of membrane-enclosed “axosomes”, axon, dendrite and spine pruning, and disassembly of synapses lead to appearance of cell-free vesicles containing cytoplasmic components of neurons, axons, neurites, spines and synapses, including proteins, RNA and their degradation products. There are other processes leading to liberation of these compounds into the extracellular medium, in particular, blebbing (Charras et al., Biophys. J. 2008, 94:1836-1853; Fackler, Grosse, J. Cell Biol. 2008, 181:879-884), exocytosis (Skog et al. Nat Cell Biol., 2008, 10:1470-1476) and other forms of active secretion.
MicroRNAs (miRNAs) are a class of non-coding RNAs whose final product is an approximately 22 nt functional RNA molecule. They play important roles in the regulation of target genes by binding to complementary regions of messenger transcripts to repress their translation or regulate degradation (Griffiths-Jones Nucleic Acids Research, 2006, 34, Database issue: D140-D144). Frequently, one miRNA can target multiple mRNAs and one mRNA can be regulated by multiple miRNAs targeting different regions of the 3′ UTR. Once bound to an mRNA, miRNA can modulate gene expression and protein production by affecting, e.g., mRNA translation and stability (e.g., Baek et al., Nature 455(7209):64 (2008); Selbach et al., Nature 455(7209):58 (2008); Ambros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116, 281-297; Cullen, 2004, Virus Research., 102, 3-9; He et al., 2004, Nat. Rev. Genet., 5, 522-531; and Ying et al., 2004, Gene, 342, 25-28). There are other classes of less characterized small RNAs (reviewed in Kim, Mol. Cells, 2005, 19: 1-15).
Many of miRNAs are specific to or over-expressed in certain organs/tissues/cells. See, e.g., Hua et al., BMC Genomics 2009, 10:214; Liang et al., BMC Genomics. 2007, 8:166; Landgraf et al., Cell. 2007, 129:1401-1414; Lee et al., RNA. 2008, 14:35-42.
Some miRNAs, including those that are cell-specific, are enriched in certain cellular compartments, particularly in axons, dendrites and synapses. See, e.g., Schratt et al., Nature. 439:283-289, 2006; Lugli et al., J. Neurochem. 106:650-661, 2008; Bicker and Schratt, J Cell Mol Med., 12:1466-1476, 2008; Smalheiser and Lugli, Neuromolecular Med. 11:133-140, 2009; Rajasethupathy, Neuron. 63:714-716, 2009; Kye, RNA 13:1224-1234, 2007; Yu et al., Exp Cell Res. 314:2618-2633, 2008; Cougot, et al., J. Neurosci. 28:13793-13804, 2008; Kawahara, Brain Nerve. 60:1437-1444, 2008; Schratt G. Rev Neurosci. 2009; 10:842-849.
Expression and concentrations of miRNAs are regulated by various physiological and pathological signals. Changes in expression of some miRNAs were found in neurons of Alzheimer's and other neurodegenerative disease patients. Hébert and De Strooper, Trends Neurosci. 32:199-206, 2009; Saba et al., PLoS One. 2008; 3:e3652; Kocerha et al., Neuromolecular Med. 2009; 11:162-172; Sethi and Lukiw, Neurosci Lett. 2009, 459:100-104; Zeng, Mol Pharmacol. 75:259-264, 2009; Cogswell et al., Journal of Alzheimer's Disease. 14: 27-41, 2008; Schaefer et al., J. Exp. Med. 204:1553-1558, 2007; Hébert, Proc Natl Acad Sci USA. 2008; 105:6415-6420; Wang et al., J. Neurosci. 2008, 28:1213-1223; Nelson et al., Brain Pathol. 2008; 18:130-138; Lukiw, Neuroreport. 2007; 18:297-300.
Due to their small size, miRNAs can cross the blood-brain, placental and kidney barriers. Analysis of cell/tissue-specific miRNAs in bodily fluids was proposed for detection of in vivo cell death (U.S. Patent Pub. No 20090081640; Laterza et al., Clin Chem. 2009, 55:1977-1983).
Cognitive function testing and brain imaging, which are currently used as main methods for diagnosis of neurodegenerative diseases such as AD, allow only detection of later stages of disease and are not sufficiently specific. There is still a great need in the art to develop methods for early diagnosis of neurodegenerative diseases and other neurological disorders in mammals prior to occurrence of major morphological changes and massive neuronal cell death.